Multi-effect repair type collagen and application thereof

Abstract

The application discloses a kind of multi-effect repair type collagen and its application in the technical field of synthetic biology, the application selects transdermal peptide TD-1, the coding gene of the transmembrane region of human XVII type collagen, extracellular sixteenth non-collagen region and extracellular fifteenth collagen region and His tag is connected in series, and gene sequence is optimized by pichia pastoris codon selection preference, then obtains T-COL17R3 by construction and expression. Through efficacy experiment, it is verified that T-COL17R3 compared with similar products on sale, not only excellent transdermal performance, also has more optimal ability of promoting cell proliferation, migration, anti early glycation product ketone amine and anti late glycation product dicarbonyl compound, better free radical scavenging ability and anti-elastase ability, and can reduce the synthesis of melanin in B16-F10 cell, therefore, T-COL17R3 has excellent application potential in the development of drug composition or skin care product with repair, anti-wrinkle firming or whitening effect.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology technology, specifically to a multi-functional repair collagen and its applications. Background Technology

[0002] The skin, the largest organ in the human body, protects us from mechanical stress and pathogens. To perform these functions, basal keratinocytes constantly detach from the basement membrane (BM), divide, and migrate upwards. The basement membrane, also known as the epidermal-dermal junction, anchors the epidermis and dermis through various extracellular matrix proteins beneath the dermis, and functions to transmit signals and nutrients, maintain skin structure and firmness, and so on. A healthy basement membrane performs a variety of biological functions, ensuring smooth circulation between the epidermis, basement membrane, and dermis, resulting in healthy and intact skin.

[0003] Type XVII collagen is a core element for the stability of the basement membrane. It combines with integrins to form hemidesmosomes, which connect type IV and type VII collagen to form a complete, supportive semi-permeable membrane structure that stabilizes the dermal-epidermal junction and maintains skin integrity. In photoaged skin, the basement membrane flattens, type XVII collagen expression decreases significantly, hemidesmosomes loosen, and skin structure and function become abnormal, manifesting as skin fragility and dysfunction. Supplementing with type XVII collagen can repair the basement membrane, regulate cell self-cleaning, and promote collagen regeneration. In the process of skin wound repair, type XVII collagen plays an important role by influencing stem cell migration, proliferation, and differentiation. With the development of synthetic biology technology, recombinant type XVII collagen can be obtained as a novel therapeutic collagen and anti-aging raw material, bringing a new breakthrough to skin anti-aging.

[0004] Compared with animal-derived collagen, recombinant humanized collagen not only has better bioactivity, biocompatibility, and water solubility, but also has a lower risk of pathogens. Type XVII collagen is scarce, and animal extraction is not feasible. With the large-scale application of genetic engineering technology, the bottleneck of large-scale preparation of type XVII collagen has been successfully solved by using recombinant expression of exogenous proteins through genetic engineering.

[0005] A prior application (application number 202510847175.4) proposed a highly transdermal recombinant humanized type XVII collagen, its preparation method, and its applications. Two recombinant humanized type XVII collagen proteins, named T-COL17-NC16 and T-COL17, were constructed by sequentially expressing the encoding gene of the transdermal peptide TD-1 (optimized by Pichia pastoris codon selection preference), different functional fragments of human type XVII collagen, and the His tag gene. Efficacy experiments verified that, compared to similar commercially available products, they not only possess excellent repair, anti-wrinkle, firming, and whitening effects but also superior transdermal absorption. This invention proposes a multi-effect repair collagen and its applications to further promote the application effects of recombinant humanized type XVII collagen in pharmaceutical compositions or skincare products. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-functional repair collagen and its applications in order to solve the above-mentioned problems.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] As a first aspect of the present invention, a multi-functional repair collagen T-COL17R3 is disclosed, comprising a transdermal peptide TD-1, a partial functional fragment of type XVII collagen, and a His tag. The partial functional fragment of type XVII collagen is selected from the transmembrane region, the extracellular sixteenth non-collagenous region, and the extracellular fifteenth collagenous region of type XVII collagen. The amino acid sequence of the multi-functional repair collagen T-COL17R3 is shown in SEQ ID NO.1.

[0009] As a second aspect of the present invention, a polynucleotide encoding the aforementioned multi-functional repair collagen T-COL17R3 is also disclosed, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0010] As a third aspect of the present invention, a recombinant plasmid is also disclosed, the recombinant plasmid comprising the polynucleotide sequence as described above, and capable of correspondingly translating and expressing the multifunctional repair collagen T-COL17R3 as described above.

[0011] As a fourth aspect of the invention, a host cell is also disclosed, the host cell comprising the recombinant plasmid as described above or expressing the multifunctional repair collagen T-COL17R3 as described above.

[0012] As a further optimization of the present invention, the host cell is Pichia pastoris.

[0013] As a fifth aspect of the present invention, a method for preparing the multi-effect repair collagen as described above is also disclosed, comprising the following steps:

[0014] (1) Inoculate the host cells as described above into the fermentation medium for fermentation culture, and after the fermentation culture is completed, centrifuge and take the supernatant to obtain the fermentation liquid;

[0015] (2) The fermentation liquid obtained in step (1) is separated and purified, and the protein is detected to obtain the multi-effect repair collagen.

[0016] As a sixth aspect of the present invention, the use of a multi-functional repair collagen as described above in the preparation of pharmaceutical compositions or skin care products is also disclosed.

[0017] As a further optimization of the present invention, the pharmaceutical composition or skin care product has at least one of the effects of repair, anti-wrinkle and firming, or whitening and brightening.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention selects the transdermal peptide TD-1, the transmembrane region of human type XVII collagen, the extracellular sixteenth non-collagenous region, the extracellular fifteenth collagenous region, and the His tag encoding gene in tandem, and optimizes the gene sequence using Pichia pastoris codon selection bias. After construction and expression, recombinant humanized type XVII collagen, namely T-COL17R3, is obtained. Furthermore, efficacy experiments have verified that T-COL17R3, compared to similar commercially available products, possesses multiple effects including repair, anti-wrinkle firming, and whitening / brightening, and exhibits excellent transdermal absorption, showing promising application prospects in pharmaceutical compositions or skincare products. Attached Figure Description

[0020] Figure 1 This test measures the ability of recombinant humanized type XVII collagen to promote cell proliferation.

[0021] Figure 2 The results show the ability of recombinant humanized type XVII collagen to promote cell migration.

[0022] Figure 3 This is a graph showing the changes in cell scratch area at different times;

[0023] Figure 4 These are the results of the cytotoxicity test of recombinant humanized type XVII collagen against 3T3 cells;

[0024] Figure 5 These are the results of the detection of the adhesion-promoting effect of recombinant humanized type XVII collagen on 3T3 cells;

[0025] Figure 6The results show the inhibition rate of recombinant humanized type XVII collagen on melanin production in B16-F10 cells.

[0026] Figure 7 This is a schematic diagram of the diffusion cell used in transdermal performance testing;

[0027] Figure 8 These are the transdermal performance test results of recombinant humanized type XVII collagen and similar commercially available products over 24 hours.

[0028] Figure 9 These are the transdermal performance test results of recombinant humanized type XVII collagen and similar commercially available products after 48 hours.

[0029] Figure 10 This is the result of the test for the anti-non-enzymatic glycosylation ability of aminoguanidine (AG);

[0030] Figure 11 This is the result of the test on the ability of recombinant humanized type XVII collagen to resist early glycation product ketamine;

[0031] Figure 12 This is the result of the test on the ability of recombinant humanized type XVII collagen to resist dicarbonyl compounds of late glycation products;

[0032] Figure 13 The results show the ability of recombinant humanized type XVII collagen to resist HACAT cell glycosylation.

[0033] Figure 14 This is the result of the test on the free radical scavenging ability of recombinant humanized type XVII collagen ABTS;

[0034] Figure 15 This is the result of the ABTS free radical scavenging ability test, which is a positive control. Detailed Implementation

[0035] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0036] Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0037] YPD liquid culture medium: 10g yeast extract, 20g trypton, 20g glycerol, ultrapure water to a final volume of 1000ml, sterilize at 121℃ for 20min;

[0038] YPD solid medium: Add 15g of agar to YPD liquid medium per 1000ml;

[0039] BMGY medium: 10g yeast extract, 20g trypton, 40g glycerol to a final volume of 800ml, add 100ml 1M phosphate buffer, sterilize at 121℃ for 20min, cool, and add 100ml filtered sterile 10×YNB and 2ml biotin.

[0040] 0.2M Phosphate Buffer (PB): Dissolve 27.22g K2HPO4 and 8g NaCl in 800ml of ultrapure water, adjust the pH to 7.4, and bring the volume to 1000ml. This is solution A.

[0041] 3M NaCl: 175.32g NaCl dissolved in 1000ml ultrapure water, this is solution B.

[0042] Nickel column chromatography Binding Buffer: 100ml solution A, 100ml solution B, add ultrapure water to 800ml, adjust pH to 7.4, and bring the volume to 1000ml.

[0043] Nickel column chromatography Wash Buffer: 100 ml solution A, 100 ml solution B, add ultrapure water to 800 ml, adjust pH to 7.4, and bring the volume to 1000 ml.

[0044] Nickel column chromatography Elution Buffer: 100ml solution A, 100ml solution B, 34g imidazole, add ultrapure water to 800ml, adjust pH to 7.4, and bring the volume to 1000ml.

[0045] 0.02M Phosphate Buffer (PB): Dissolve 2.4g NaH2PO4 and 2.8g Na2HPO4 in 800ml of ultrapure water, adjust the pH to 7.4, and bring the volume to 1000ml.

[0046] 1. Gene design and synthesis

[0047] 1.1 Design and synthesis of the gene encoding T-COL17R3

[0048] The transmembrane region of transdermal peptide TD-1, the sixteenth extracellular non-collagenous region, and the largest extracellular fifteenth collagenous region were selected and tandemly linked with the His tag coding gene. The gene sequence was optimized according to the codon selection preference of Pichia pastoris to obtain the target gene sequence. Then, the optimized full gene sequence was obtained through whole-genome synthesis (synthesized by Genewiz). The full gene sequence is shown in SEQ ID NO.3, and the encoded collagen is T-COL17R3, whose amino acid sequence is shown in SEQ ID NO.1.

[0049] 1.2 Construction of recombinant expression engineered bacteria

[0050] The plasmid and the constitutive vector plasmid pGAPZα were double-digested with enzymes, and the target fragment and the pGAPZα expression vector fragment were obtained by gel extraction. The target fragment and the expression vector were ligated using a ligase, and the ligation product was transformed into competent E. coli TOP10. Positive clones were screened on LB resistant plates containing Zeocin, and the recombinant constitutive expression plasmid was successfully obtained.

[0051] The recombinant constitutive expression plasmid was linearized and electroporated into competent Pichia pastoris X33 cells. The glyceraldehyde-3-phosphate dehydrogenase promoter in X33 cells eliminates the need for methanol during fermentation, avoiding the pollution and hazards caused by large-scale methanol use in production, making it more suitable for large-scale production. Using Zeocin as an antibiotic marker and his tag antibody, Dot-Blot and Western-Blot analyses were performed to obtain the recombinant Pichia pastoris engineered strain.

[0052] 1.3 Fermentation Expression

[0053] Recombinant Pichia pastoris was streaked onto YPD solid agar plates and incubated at 30°C until single colonies appeared. A single colony was picked and inoculated into 10 ml of YPD liquid agar, and cultured overnight at 30°C with shaking at 220 rpm to obtain the primary seed culture. 1 ml of the primary seed culture was added to 200 ml of BMGY medium and incubated at 30°C with shaking at 220 rpm for 24 hours to obtain the secondary seed culture. The secondary seed culture was then added to 4 L of BMGY liquid agar and incubated at 30°C with shaking at 220 rpm for 60 hours. The supernatant was collected by centrifugation for subsequent separation and purification experiments.

[0054] 1.4 Separation and Purification

[0055] T-COL17-R3 is expressed intracellularly due to the presence of a transmembrane region. Intracellular proteins were extracted by cleavage with snail enzyme for subsequent separation and purification experiments.

[0056] 1.4.1 Snail enzyme lysis and extraction of intracellular proteins

[0057] Take the fermentation broth, centrifuge at 6000 rpm for 5 min, collect the cells and weigh the wet weight. Dissolve the snailase in SE buffer solution to prepare a concentration of 40 mg / ml. Add 5 ml of isotonic sorbitol solution (pH 5.8~7.2), 1 ml of snailase, and 20 μl of β-mercaptoethanol per g of cells. Incubate at 37℃ for 2 h, centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the precipitate. Resuspend with an appropriate amount of sorbitol, centrifuge at 8000 rpm for 10 min again, discard the supernatant, collect the precipitate, and repeat the washing once. Resuspend with an appropriate amount of ddH2O, freeze at -80℃ for 30 min, and then thaw at room temperature. Repeat this step 3 times. Centrifuge the lysed solution at 12000 rpm for 5 min, and collect the supernatant as yeast protein solution for subsequent experimental analysis.

[0058] 1.4.2 Obtaining the supernatant sample

[0059] The obtained supernatant was filtered through a 0.22 μm filter membrane to obtain flow-through liquid, and the pH was adjusted to 7.4 with ammonia water to obtain the supernatant sample.

[0060] 1.4.3 Nickel column affinity chromatography

[0061] Rinse the nickel column with 5-10 column volumes of ultrapure water to remove the 20% ethanol from the column (stop when the UV value remains unchanged or fluctuates around a certain value); add 10 column volumes of Binding Buffer to equilibrate the column (the Binding Buffer and supernatant should be placed on ice to minimize protein degradation loss); add the supernatant to the column and control the flow rate at 5 ml / min to collect the flow-through; then add 10 column volumes of Wash Buffer to wash the column (to elute contaminating proteins) to obtain the eluent; then elute the protein with Elution Buffer to obtain the eluent. Start collecting when the UV280 value rises sharply and stop collecting when the value decreases slowly to obtain the crude protein extract.

[0062] 1.4.4 Desalination using a desalination column

[0063] The crude protein extract was loaded onto a desalting column, and the washing buffer was PB. Collection began when the UV280 increased and stopped when the conductivity increased to obtain recombinant humanized type XVII collagen T-COL17R3. The protein concentration was detected by BCA method, and the purity of the collected protein was detected by SDS-PAGE.

[0064] 2. Efficacy Test

[0065] To further illustrate the efficacy of the T-COL17R3 synthesized in this invention, in the following efficacy tests, commercially available recombinant humanized type XVII collagen and T-COL17-NC16 disclosed in the prior application (application number 202510847175.4) were used as controls.

[0066] Among them, the commercially available recombinant humanized type XVII collagen was prepared in the form of recombinant expression of exogenous protein by the above-mentioned genetic engineering, and its amino acid sequence was obtained by repeating the sequence shown in SEQ ID NO.2 three times.

[0067] 2.1 Repair Efficacy Test

[0068] 2.1.1 Detection of cell proliferation promotion capacity

[0069] Take a 96-well plate and add the prepared HACAT cell suspension (purchased from Nanjing Kebai Biotechnology Co., Ltd.) to a density of 10,000 cells / well. Fill the edge wells with sterile PBS. Incubate the seeded cell culture plate in an incubator for about 24 hours until the cells are completely adhered and grow stably. Remove the plate, discard the cell culture medium, and add 100 µl of the drug at a concentration gradient, setting 6 replicates. Incubate at 37°C with 5% CO2 for 24 hours or other suitable time (calculated based on the drug administration time). Observe the drug effect under an inverted microscope. Discard the culture medium, wash each well twice with sterile PBS, add 100 µl of culture medium and 10 µl of CCK-8 to each well, incubate at 37°C for about 1 hour, and measure the absorbance at 450 nm using a microplate reader. Use the value of the negative control group as a baseline to calculate the relative cell proliferation rate of each experimental group.

[0070] Relative cell proliferation rate = (OD) 实验组 / OD 对照组 ) × 100%.

[0071] The results are as follows Figure 1 As shown, T-COL17R3 has a good ability to promote cell proliferation, comparable to T-COL17-NC16. At a concentration of 40 μg / ml, T-COL17R3 promotes cell proliferation by nearly 1.2 times that of the negative control group.

[0072] 2.1.2 Detection of cell migration promotion ability

[0073] HACAT cells were seeded into 12-well plates at a seeding density of 200,000 cells / well and cultured in DMEM medium containing 10% FBS until confluence. A crisscross pattern was created in each well of the confluent 12-well plate using a 1 ml sterile pipette tip. Exfoliated cells were washed away with PBS, and fresh medium and medication were added to each well (1 ml). Cells were observed and photographed at 0, 6, 10, and 24 hours post-scribing, and cell migration was calculated using the following formula.

[0074] Cell migration rate = {(initial scratch area - scratch area at time t) / initial scratch area} × 100%.

[0075] The results are shown in Table 1. Figure 2 and Figure 3 As shown.

[0076] Table 1. Results of the cell migration-promoting ability of recombinant humanized type XVII collagen.

[0077] The results showed that T-COL17R3 at a concentration of 5 μg / ml had a significant cell migration-promoting effect after 24 hours of treatment, with an average cell migration rate of 100%, which was significantly better than T-COL17-NC16 and similar commercially available products.

[0078] 2.1.3. Effect on adhesion promotion of 3T3 cells

[0079] (1) Cytotoxicity test against 3T3 cells

[0080] When the adherent cells have grown to approximately 80%–90% confluence, remove the culture supernatant, trypsinize the cells to obtain a single-cell suspension according to the cell passage method, aspirate 50 μL of the cell suspension for cell counting, and adjust the cell density to 1 × 10⁻⁶ cells / cells with complete culture medium. 5 The cell count was approximately [number] cells / mL. A 96-well plate was used for the experiment, with 100 μL of cell suspension seeded into each well. Six parallel wells were prepared, and sterile PBS was added to the edge wells to prevent evaporation. The seeded cell culture plates were placed in an incubator for pre-culture for 24 hours. The culture supernatant was removed, and 100 µL of different concentrations of the test drug were added. The plates were then incubated in the cell culture incubator for another 24 hours or other suitable time, calculated based on the drug administration time and adjusted according to experimental requirements. Cell morphology was observed under a microscope. Cells were washed twice with PBS, and 100 µL of LDM medium and 10 µL of CCK8 reagent were added to each well. The plates were incubated at 37°C for approximately 60 minutes, and the absorbance at 450 nm was measured using a microplate reader. The relative cell proliferation rate was calculated based on the absorbance.

[0081] Relative cell proliferation rate = (OD) 实验组 / OD 对照组 ) × 100%.

[0082] The results are as follows Figure 4 As shown, a concentration of 10 μg / ml showed no significant cytotoxicity to 3T3 cells, and this concentration can be used in subsequent adhesion-promoting experiments.

[0083] (2) Adhesion assay of 3T3 cells

[0084] Dermal fibroblasts secrete extracellular matrix components such as collagen and elastin to maintain the mechanical properties of the skin. Decreased adhesion leads to downregulation of type I and type III collagen gene expression, causing the extracellular matrix structure to collapse, resulting in gradual skin laxity and deepening wrinkles. In addition, the migration ability of fibroblasts with weakened adhesion also gradually declines, leading to delayed wound healing. This phenomenon is more common in elderly skin.

[0085] This invention uses the NIH / 3T3 mouse fibroblast model to systematically evaluate the cytotoxicity and adhesion-promoting activity of recombinant human type XVII collagen on fibroblasts. The specific steps are as follows:

[0086] The sample was diluted to 10 μg / mL artificial basement membrane gel using DMEM medium without fetal bovine serum, mixed well, and 100 μL was added to each well of a 96-well plate. The plate was then incubated overnight at 4°C.

[0087] Remove the coating solution and wash the well plate three times with serum-free medium.

[0088] Single-cell suspensions were prepared using cell passage. Cells were collected by centrifugation for 3 minutes at 300g, resuspended in serum-free DMEM medium, and the cell density was adjusted to 5 × 10⁶ cells / mL. 5 cells / mL;

[0089] 100 μL was seeded into each well of a 96-well plate, and experimental, control, blank, and standard groups were set up respectively.

[0090] Experimental group: collagen basement membrane + test cells;

[0091] Control group: Collagen basement membrane + serum-free culture medium;

[0092] Blank group: Serum-free culture medium containing the cells to be tested;

[0093] Standard group: serum-free culture medium;

[0094] The experimental group had 6 replicates, and the other groups had 3 replicates each. The edge wells were filled with sterile PBS buffer.

[0095] Culture the cells at 37°C and 5% CO2 for 2 hours (the culture time can be adjusted appropriately according to the cell condition and experimental requirements).

[0096] Remove the supernatant (no operation is required for the blank and standard groups), wash 3 times with PBS, and add 100 μL of fresh culture medium and 10 μL of CCK-8 detection solution to each well (add 10 μL of CCK-8 detection solution directly to the blank and standard groups).

[0097] The absorbance of each sample well at a wavelength of 450 nm was measured using an ELISA reader, and the detection data were recorded and saved.

[0098] Calculate cell adhesion rate: Cell adhesion rate = [(Experimental group cell OD - Control group OD) / (Blank group cell OD - Standard group OD)] × 100%

[0099] All operations must be performed under sterile conditions; air bubbles should be avoided during the coating process; the cell suspension must be thoroughly mixed before cell inoculation.

[0100] The results are as follows Figure 5 As shown, in cell adhesion experiments, T-COL17R3 with added transmembrane segments exhibited superior adhesion-promoting effects compared to T-COL17-NC16 and similar commercially available products. T-COL17R3 can promote cell attachment and anchoring on the ECM, providing favorable conditions for the construction of the extracellular microenvironment.

[0101] 2.2 Whitening and Brightening Efficacy Test

[0102] 2.2.1 Effect of inhibiting melanin activity

[0103] Mouse melanoma cells B16 F10 (purchased from ATCC, model CRL-6475) can synthesize and secrete melanin. Melanin is an amino acid derivative that reacts with sodium hydroxide to produce a water-soluble compound. The melanin content was calculated by detecting the ultraviolet absorbance at 405 nm. Mouse melanoma cells B16 F10 were treated with melanocyte-stimulating hormone (αMSH). The inhibition of melanin production was calculated and determined by comparing the melanin secreted by these cells with that of cells in the sample group, thus evaluating the skin-whitening activity.

[0104] The detection method is as follows:

[0105] First, we examined the effect of recombinant humanized type XVII collagen on the proliferation of melanoma cells B16-F10, and then selected the concentration that had no effect on the proliferation of B16-F10 cells for subsequent experiments.

[0106] Take a 6-well plate and add 2 ml of B16-F10 cell suspension to each well to achieve a cell density of 4 × 10⁻⁶ cells / well. 4Cells / well were collected and incubated in a 5% CO2 incubator for 24 h. The supernatant was discarded. According to the sample loading information in Table 5, 2 ml of solution was added to each well, designated as the blank control group, model group, positive control group, and experimental group, respectively. The cells were then incubated in a 5% CO2 incubator at 37°C for 72 h (based on drug administration time). The supernatant was discarded. Samples were then added again according to Table 2, with 2 ml of DMEM complete medium added to each well. Incubate at 37°C in a CO2 incubator for 48-72 hours, until the cell confluence rate reaches over 90% as observed under a microscope. Then discard the culture supernatant and wash each well twice with 1 ml of sterile PBS buffer. Discard the PBS buffer and add 200 μl of trypsin solution to each well for 3 min to digest. Add 1 ml of PBS buffer to each well and gently pipette the digested cells. Transfer the cell suspension to a 1.5 ml EP tube and centrifuge at 300 g for 5 min, discarding the supernatant. Add 150 μl of melanin extraction solution to each tube and incubate at 90°C for 1 hour to completely dissolve the melanin. Transfer 100 μl of the solution from each EP tube to a 96-well plate. Using the melanin extraction solution as a zeroing control, measure the absorbance at 405 nm using a microplate reader. Express the melanin content as OD405 and calculate the melanin synthesis inhibition rate using the following formula: ;

[0107] Table 2 Sample Addition Table

[0108] The results are as follows Figure 6 As shown, T-COL17R3 can reduce melanin synthesis in B16-F10 cells, and the inhibitory effect increases with increasing concentration. When the concentration is increased from 10 μg / ml to 20 μg / ml, the inhibitory effect on melanin is significantly improved. In addition, at the same concentration, the inhibitory effect of T-COL17R3 on melanin is better than that of T-COL17-NC16.

[0109] 2.2.2 Transdermal Performance Test of Recombinant Humanized Type XVII Collagen

[0110] To assess the transdermal absorption capacity of recombinant humanized type XVII collagen, this study employed a static diffusion cell method to determine its transdermal absorption capacity, comparing it with commercially available similar products as a positive control group. The specific steps were as follows:

[0111] Take the skin of a 1-month-old Bama pig and fix it in place, with the stratum corneum facing upwards and the dermis downwards for each skin section. Install the diffusion pool (see the diagram of the diffusion pool). Figure 7As shown in the figure, the pig skin sample was kept in contact with the liquid surface of the receiving pool; 6.5 ml of PBS was added to the receiving chamber, and 1 ml of each group of solutions shown in Table 3 was added to the supply chamber; the diffusion instrument was started, and the duration was set to 24 h and 48 h for two groups, with three parallel samples in each group; the collagen content in the receiving pool at each time point was detected by BCA method.

[0112] Table 3 Solution information for each group

[0113] ;

[0114] from Figure 8 , Figure 9 As can be seen, in the experimental group, the content of recombinant humanized type XVII collagen T-COL17R3 in the receiving pool after 24h and 48h was significantly higher than that of T-COL17-NC16 and similar commercially available products. This shows that the transdermal transdermal ability of recombinant humanized type XVII collagen T-COL17R3 provided by the present invention is significantly improved compared with T-COL17-NC16 and similar commercially available products.

[0115] 2.3 Anti-wrinkle and firming efficacy test

[0116] 2.3.1 Detection of resistance to non-enzymatic glycosylation

[0117] The NBT method was used to determine the content of ketamines in early glycation products, and the Girard-T method was used to determine the content of dicarbonyl compounds in late glycation products. The specific steps are as follows:

[0118] (1) Solution preparation

[0119] MGO stock solution: Prepare a 40mM solution with ultrapure water and store at 5℃±3℃.

[0120] BSA stock solution: Prepare a 20 mg / ml solution with ultrapure water and store at 5℃±3℃.

[0121] (MGO+BSA) working solution: Accurately measure MGO (40mM) and BSA (20mg / ml) in a 1:1 ratio to prepare 20mM MGO + 10mg / ml BSA. Prepare fresh before use.

[0122] NBT working solution: Weigh NBT and prepare a 0.3 mM NBT solution using a buffer solution with a pH of 10.35.

[0123] Girard-T working solution: Weigh Girard-T and prepare a 500mM Girard-T solution with ultrapure water.

[0124] Sodium formate working solution: Weigh out sodium formate solid and prepare a 500mM sodium formate solution using acetate buffer solution with a pH of 3.7.

[0125] Positive control AG (aminoguanidine) stock solution: Weigh an appropriate amount of AG, add ultrapure water to prepare 5 mg / ml solution, and store at 5℃±3℃.

[0126] Positive control AG working solution: Accurately measure an appropriate amount of AG stock solution (5 mg / ml) and prepare a series of concentration gradient solutions at 1000 ug / ml, 500 ug / ml, 250 ug / ml, 125 ug / ml, and 62.5 ug / ml to verify the system.

[0127] (2) Prepare the reaction system according to Table 4-5, and test after reacting at 37℃ for 5 days.

[0128] Table 4. Experimental Sample Addition Table (ml)

[0129] ;

[0130] Table 5. Experimental Sample Addition Table (ml)

[0131] The results are as follows Figure 10 , Figure 11 and Figure 12 As shown, T-COL17R3 and T-COL17-NC16 have comparable abilities to resist early glycation products ketamine and late glycation products dicarbonyl compounds, and their abilities far exceed those of commercially available similar products at the same concentration.

[0132] 2.3.2 Test of anti-HACAT cell glycosylation ability

[0133] To further verify the anti-non-enzymatic glycosylation activity of the recombinant protein, this invention induced non-enzymatic glycosylation in keratinocytes with methylglyoxal (MGO), thereby constructing a cellular-level non-enzymatic glycosylation model. The specific steps are as follows:

[0134] (1) After confirming under a microscope that the cell confluence has reached 80%~90%, remove the culture supernatant;

[0135] (2) Cells were processed according to the cell passage method to obtain a single-cell suspension. 100 μL of the cell suspension was used for cell counting, and the cell density was adjusted to 1 × 10⁻⁶ cells / cells with complete culture medium. 5 cells / mL;

[0136] (3) Seed the adjusted cell suspension at 100 μL / well in a 96-well plate, and add sterile PBS buffer to the edge wells to prevent evaporation;

[0137] (4) Place the inoculated cell culture plate in a constant temperature incubator and incubate for about 24 hours;

[0138] (5) Discard the culture supernatant, add the MGO inducer solution prepared by DMEM, and continue to incubate in a 5% CO2, 37℃ incubator for 24 hours;

[0139] (6) Remove the inducing agent solution and add serum-free culture medium containing different concentrations of type XVII collagen and positive control AG respectively. Incubate at 37°C for 48 hours.

[0140] (7) Observe the changes in cell morphology under a microscope, wash the cells twice with sterile PBS, add 100uL of culture medium and 10uL of CCK-8 solution to each well, incubate at 37℃ for 60 minutes, and detect the absorbance at 450nm with an ELISA reader.

[0141] The results are as follows Figure 13 As shown, in the cell model, the cell proliferation rate in the methylglyoxal-induced group decreased to 55.24%, significantly lower than that in the group without MGO, indicating that MGO successfully induced cell damage, and the cell model was successfully constructed. Based on this model, the anti-non-enzymatic glycosylation capabilities of the two recombinant collagens at the cellular level were further explored. The experimental results showed that both recombinant proteins exhibited significant cytoprotective effects, effectively inhibiting non-enzymatic glycosylation reactions in cells, thereby delaying skin aging and improving skin wrinkles and sagging. At a concentration of 10 μg / mL, the anti-glycosylation effect of T-COL17R3 was superior to that of commercially available similar products, but inferior to that of T-COL17-NC16.

[0142] 2.3.3 Antioxidant Activity Test

[0143] Take 50 μl each of recombinant humanized type XVII collagen at different concentrations, positive control Trolox (water-soluble vitamin E derivative), and commercially available similar products, add 150 μl of ABTS working solution (0.74 mmol / L ABTS, 0.26 mmol / L K2S2O8), incubate at room temperature for 6 min, and measure absorbance at 734 nm. Calculate the scavenging effect of the samples on ATBS free radicals and plot the scavenging rate curve.

[0144] The results are as follows Figure 14 and Figure 15 As shown, T-COL17R3 and T-COL17-NC16 exhibit comparable free radical scavenging capabilities, which are concentration-dependent and significantly higher than those of similar commercially available products. This indicates that the T-COL17R3 proposed in this invention has potential anti-wrinkle and firming effects.

[0145] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A multi-functional repair collagen, characterized in that, The multi-effect repair collagen T-COL17R3 includes a transdermal peptide TD-1, a transmembrane region of type XVII collagen, an extracellular sixteenth non-collagen region, a large extracellular fifteenth collagen region, and a His tag. The amino acid sequence of the multi-effect repair collagen T-COL17R3 is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that, The nucleotide sequence of the multi-functional repair collagen T-COL17R3 as described in claim 1 is shown in SEQ ID NO.

3.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the polynucleotides as described in claim 2 and is capable of correspondingly translating and expressing the multifunctional repair collagen T-COL17R3 as described in claim 1.

4. A host cell, characterized in that, The host cell contains the recombinant plasmid as described in claim 3 or expresses the multifunctional repair collagen T-COL17R3 as described in claim 1.

5. A host cell according to claim 4, characterized in that, The cells include Pichia pastoris.

6. A method for preparing the multi-effect repair collagen as described in claim 1, characterized in that, Includes the following steps: (1) The host cells as described in any one of claims 4-5 are inoculated into a fermentation medium for fermentation culture. After the fermentation culture is completed, the supernatant is collected by centrifugation to obtain the fermentation liquid. (2) The fermentation liquid obtained in step (1) is separated and purified, and the protein is detected to obtain the multi-effect repair collagen.

7. The application of the multi-functional repair collagen as described in claim 1 in the preparation of skin care products.

8. The application according to claim 7, characterized in that, The skincare product has at least one of the following effects: repair, anti-wrinkle and firming, or whitening and brightening.

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